This project is aimed at a greater understanding of the mechanisms of regulation of neurotransmitter release, which is critical to an understanding of the function of the nervous system in health and disease. It uses a novel vertebrate preparation: the synapses formed by motorneuron neurites on muscle cells in Xenopus nerve muscle cultures, in which pre- and postsynaptic processes can be patched-clamped and ionic currents analyzed directly and correlated with quantal neurotransmitter release. Of particular interest is the mechanism whereby Ca2+ influx triggers vesicle fusion. Ca2+ dynamics at active zones has been modeled extensively, but direct measurements have proved difficult. In the Xenopus synapses, large conductance Ca2+-dependent K+ (BK or rnaxi-K) channels are functionally coupled to Ca2+channels and concentrated at active zones. The I<BK> activation rate is proportional to the instantaneous local Ca2+ concentration. These endogenous channels can be used to measure accurately the Ca2+ concentration at the active zone. We will quantify the changes in Ca2+ concentration that occur at active zones during step depolarizations, action potentials of differing shape, and other stimulus waveforms that allow a controlled change in Ca2+ concentration at the active zone over the range of about 200 nM to more than 100 muM. We will explore the roles of endogenous fixed and mobile Ca2+ buffers in modulating the build-up and decay of Ca2+ "microdomains," and the possible roles of endogenous Ca2+ sequestering compartments and Ca2+-induced Ca2+ release in regulating Ca2+ dynamics. All of these changes in Ca2+ concentration and dynamics will be correlated with levels and timing of neurotransmitter release. We will fit data describing I/BK behavior and transmitter release into a mathematical model of Ca2+ microdomains and their interactions with BK channels and release sensors. A major component of the project is aimed at understanding the mechanisms of short-term synaptic plasticity, in particular, testing the hypothesis that facilitation of release is the result of partial saturation of endogenous buffers that suppress the peak build-up of Ca2+ to an initial impulse, but because of continued binding, are unable to buffer a second I/Ca as effectively.